Discovery and structural characterization of monkeypox virus methyltransferase VP39 inhibitors reveal similarities to SARS-CoV-2 nsp14 methyltransferase

Monkeypox is a disease with pandemic potential. It is caused by the monkeypox virus (MPXV), a double-stranded DNA virus from the Poxviridae family, that replicates in the cytoplasm and must encode for its own RNA processing machinery including the capping machinery. Here, we present crystal structures of its 2′-O-RNA methyltransferase (MTase) VP39 in complex with the pan-MTase inhibitor sinefungin and a series of inhibitors that were discovered based on it. A comparison of this 2′-O-RNA MTase with enzymes from unrelated single-stranded RNA viruses (SARS-CoV-2 and Zika) reveals a conserved sinefungin binding mode, implicating that a single inhibitor could be used against unrelated viral families. Indeed, several of our inhibitors such as TO507 also inhibit the coronaviral nsp14 MTase.

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Crystallography: Diffraction data was collected from a single frozen crystal for each dataset. Crystallographic statistics was calculated within each dataset and was summarized in Table 1. Enzymatic assays: Figure 4: The inhibition curves and IC50s were based on two independent experiments (n=2). Each curve was calculated from 7 independent data points. Supplementary Figure 1: Enzymatic activities were determined using 3 independent samples (n=3); all samples were measured in technical triplicates (which were averaged to give one point in the graph).
Crystallography: Data are usually excluded when CC1/2 is lower than 0.5. Enzymatic assays: No data was excluded.
Crystallography: Redundancy was greater than 5 for each dataset and was specified in Table 1. Enzymatic assays: Figure 4: The inhibition curves and IC50s were based on two independent experiments (n=2). Each curve was calculated from 7 independent data points. Supplementary Figure 1: Enzymatic activities were determined using 3 independent samples (n=3); all samples were measured in technical triplicates (which were averaged to give one point in the graph).
Crystallography: 5% of randomly chosen reflections were excluded from calculation of final model and used for model validation and calculation of Rfree statistics. Enzymatic assays: No randomization was used.

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March 2021
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